The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na(+)/H(+) exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase C beta 1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2 PTH1R complex markedly activates phospholipase C beta and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (G(i/o) proteins). NHERF-mediated assembly of PTH1R and phospholipase C beta is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.

The stimulative effect of glucocorticoids on intestinal salt and water absorption has been known for more than two decades. However, molecular mechanisms underlying this activation remain elusive. Previous studies showed that methylprednisolone specifically increased Na ؉ /H ؉ exchanger isoform (NHE) 3 mRNA in ileum and kidney without affecting NHE1 mRNA levels. These results suggest that glucocorticoids activate NHE3 activity by induction of NHE3 transcripts. We recently found in PS120 and opossum kidney cells that chronic incubation with dexamethasone activated NHE3 independent of gene induction, indicating that the transcriptional activation may not be the only determining factor in the NHE3 activation. Furthermore, dexamethasone activated NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2, which was previously shown to confer cAMP-dependent inhibition of NHE3. This activation of NHE3 could not be duplicated by NHERF1. We identified serum-and glucocorticoid-induced protein kinase, SGK1, as the protein interacting with PDZ domains of NHERF2 to regulate NHE3 activity. The expression of SGK1 enhanced NHE3 transport in PS120 fibroblasts. In addition, the "kinase-dead" SGK1 blocked activation of NHE3 by dexamethasone in opossum kidney cells. These data demonstrated that glucocorticoid activation of NHE3 requires the activation of SGK1 and the presence of NHERF2 acting as a scaffold protein.

In the present work, we studied LPA2-mediated signaling using human colon cancer cell lines, which predominantly express LPA2. LPA2 activated Akt and Erk1/2 in response to LPA. LPA mediated Akt activation was inhibited by pertussis toxin (PTX), whereas Erk1/2 activation was completely inhibited by a blocker of phospholipase C␤, U-73122. LPA also induced interleukin-8 (IL-8) synthesis in the colon cancer cells by primarily activating LPA2 receptor. We also found that LPA2 interacts with Na ϩ /H ϩ exchanger regulatory factor 2 (NHERF2). Activation of Akt and Erk1/2 was significantly attenuated by silencing of NHERF2 expression by RNA interference, suggesting a pivotal role of NHERF2 in LPA2-mediated signaling. We found that expression of LPA2 was elevated, whereas expression of LPA1 downregulated in several types of cancers, including ovarian and colon cancer. We conclude that LPA2 is the major LPA receptor in colon cancer cells and cellular signals by LPA2 are largely mediated through its ability to interact with NHERF2.

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na + /H + exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO 3 -secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO 3 -secretory rates and blocked β 2 -adrenergic receptor (β 2 -AR) stimulation. Conversely, Nherf2 -/-mice displayed augmented FSK-stimulated HCO 3 -secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO 3 -secretion in WT mice, this effect was lost in Nherf2 -/-mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO 3 -secretion. In addition, laser microdissection and quantitative PCR revealed that the β 2 -AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the β 2 -AR and CFTR was reduced in the Nherf1 -/-mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO 3 -secretion: while NHERF1 is an obligatory linker for β 2 -AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linkingprotein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.PDZ (PSD95, Dlg, ZO-1) proteins serve as central organizers of protein complexes. The 80 -90-amino acid PDZ domains are most noted for their capacity to bind the COOH-tail of specific integral membrane proteins (1). Through a distinct interface, some PDZ domains also have the capacity to participate in separate PDZ-PDZ interactions. Interestingly, these properties allow a single PDZ domain to bind concurrently with both the COOH-tail of an integral membrane protein and another protein through a PDZ-PDZ interaction (2). PDZ proteins amplify their capacity and diversity of protein-protein interactions through co-expression of additional protein binding motifs (e.g. SH2 domains, SH3 domains, ankyrin repeats, ERM 1 binding domains), expression of multiple PDZ domains within the same PDZ protein, and oligomerization with other PDZ proteins (2-7). Oligomerization can occur either through non-PDZ domain interactions or through PDZ-PDZ interactions.ERM-binding phosphoprotein 50 (EBP50; also known as NHE regulatory factor) is a PDZ domain protein found in several epithelial cell types. EBP50 has been shown to bind a growing number of integral membrane proteins including cAMP-regulated Na ϩ /H ϩ exchanger 3 (NHE3), ␤ 2 -adrenergic receptor, G protein-coupled receptor kinase-6A (GRK6A), Yesassociated protein 65 (YAP65), and the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl Ϫ channel (cftr) (8 -12). With only two PDZ domains, individual EBP50 proteins have a limited capacity to form multiprotein arrays. Theoretically, EBP50 oligomerizatio...

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be alpha-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

Intestinal electroneutral NaCl absorption is mediated by parallel operation of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange in the enterocyte apical membrane. The ion transporters involved are Na(+)/H(+) exchanger 3 (NHE3) and the down regulated in adenoma (dra) gene product. cAMP-mediated inhibition of NHE3 requires the transporter to bind to the second PDZ (PSD95, disk large, ZO1) domain of the adapter protein NHE3 kinase A regulatory protein (E3KARP). Because the C-terminal four amino acids of dra are ETKF (glutamate-threonine-lysine-phenylalanine), resembling a PDZ interaction motif, we hypothesized that dra may also bind to one of the PDZ domains of E3KARP. In vitro the ETKF motif of dra binds to the second PDZ domain of E3KARP, the affinity being comparable to that of the known ligand CFTR. The C-terminal phenylalanine, which is an unconventional residue in PDZ interaction motifs, can only be substituted by the classical residue leucine, but not by other hydrophobic residues (valine, isoleucine). Immunofluorescence colocalizes dra, NHE3, and E3KARP in the apical compartment of human proximal colon. We suggest a model in which both NHE3 and dra bind to the second PDZ domain of E3KARP and that linking of the transporters occurs through dimerization of E3KARP. In such a model, the first PDZ domain would remain available for instance for signal transduction proteins.

The mammalian kidney plays an essential role in the control of systemic water, ion, and acid-base balance. Na ϩ /H ϩ exchanger type 3 (NHE3) plays a pivotal role in salt and fluid reabsorption in the proximal tubule (1), accounting for ϳ50% of NaCl and 70% of NaHCO 3 reabsorbed from the glomerular filtrate (2). In fact, mice deficient in NHE3 expression are relatively hypotensive, even when NHE3 in the intestine is rescued by transgenic expression (3).The renin-angiotensin system is critically involved in regulation of body blood pressure and fluid balance. The kidney secretes renin when blood pressure is low that stimulates the production of ANG 3 I, which is subsequently converted into ANG II by angiotensin-converting enzyme. The presence of ANG II receptor on the membrane of renal proximal tubules was first reported in the 1980s (4). Through the binding with its cognate receptor(s), ANG II is importantly involved in fluid reabsorption in the proximal tubules. Substantial earlier evidence from Cogan and co-workers (5-8) has demonstrated that ANG II is a potent agonist of H ϩ secretion and HCO 3 Ϫ absorption in rat proximal tubules through mechanisms dependent on decrease of cAMP, increase of [Ca 2ϩ ] i , and activation of protein kinase C (PKC). It was later shown that ANG II, at low concentrations, stimulates NHE3 activity in renal proximal tubule cells (9 -11). Studies have implicated the roles of PKC and c-Src in ANG II-mediated activation of NHE3 (9, 12). In addition, ANG II stimulates NHE3 activity through increased exocytotic insertion of NHE3 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner (13). Nevertheless, the molecular mechanisms underlying NHE3 activation by ANG II remain incompletely delineated.IRBIT was initially identified as an IP 3 receptor-binding protein and was shown to be a competitive inhibitor of Ca 2ϩ release by IP 3 receptor (14,15). We have recently identified IRBIT as a novel NHE3-interacting protein by yeast two-hybrid screening of a kidney library (16). Our study demonstrated that IRBIT binds the C-terminal domain of NHE3 and activates NHE3 activity in response to thapsigargin or ionomycin-induced rise of [Ca 2ϩ ] i in PS120 fibroblast cells (16). IRBIT mRNA is ubiquitously present in all tissues, but the highest expression was reported in the brain, reproductive tissues, and kidney (14). These findings prompted us to hypothesize that IRBIT might play an important role in NHE3 regulation in the kidney. In this work, we investigated the role of IRBIT in the regulation of NHE3 by ANG II. Our findings show that IRBIT is critically involved in the activation of NHE3 by ANG II, and this regulation is Ca 2ϩ -CaMKII-dependent.